MCF7 cells (0.4 × 106 cells) were seeded in growth medium on 6-well culture plates for 1 day. The cells were serum starved in DMEM/F-12 medium (Gibco) for 20 h. After serum starvation, the cells were co-stimulated with human IgG, M30103, B30104, or A12 (1–10 μM) and recombinant human IGF-1 (200 ng/mL, R&D systems, 291-G1) for 20 min. The cells were lysed in mammalian protein extraction reagent (M-PER; Fisher Scientific, 78501) with protease and phosphatase inhibitor cocktail (PIC; Pierce, 1861281). The cell lysates were separated on 4–12% Bis-Tris gels and transferred to PVDF membranes. Finally, the proteins were blotted with antibodies against phosphorylated-Akt (Cell Signaling, 9271S), total Akt (Cell Signaling, 9272S), phosphorylated-IGF1R (Cell Signaling, 3024S), total IGF1R (Cell Signaling, 3018S), and β-actin (Santa Cruz, Sc-47778).
Phosphorylated igf1r
Manufactured by Cell Signaling Technology
The Phosphorylated-IGF1R is a laboratory reagent that detects the phosphorylated form of the Insulin-like Growth Factor 1 Receptor (IGF1R) protein. This product is designed to facilitate the study of IGF1R activation and signaling pathways in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Total igf1r, by Cell Signaling Technology (1 mentions)
M per, by Thermo Fisher Scientific (1 mentions)
Recombinant human igf 1, by R&D Systems (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Phosphorylated akt, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Mounting solution, by Vector Laboratories (1 mentions)
Diaminobenzidine dab detection reagent, by Enzo Life Sciences (1 mentions)
Dako protein block, by Agilent Technologies (1 mentions)
Avidin biotin complexes, by Vector Laboratories (1 mentions)
Phosphorylated src, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
2 protocols using phosphorylated igf1r
1
Modulation of IGF-1 signaling in MCF7 cells
2
Immunohistochemical Analysis of Murine Lung Tissues
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Sections derived from formalin-fixed and paraffin-embedded murine lung tissues were deparaffinized by incubation overnight at 65 °C followed by rehydration in sequential xylene and ethanol rinses. After incubation with hydrogen peroxide, the slides were washed with PBS and then incubated with 0.4% Triton X-100. The sections were incubated with blocking solution (Dako Protein Block, Dako, Glostrup, Denmark) for 30 min at room temperature after washing with PBS. The sections were further incubated with primary antibodies (phosphorylated IGF1R, phosphorylated Src, and cleaved caspase 3 [all from Cell Signaling], diluted at 1:200) overnight at 4 °C, washed with PBS several times, incubated with the corresponding biotinylated secondary antibodies (diluted at 1:500), and then washed with PBS multiple times. After adding avidin-biotin complexes (Vector Laboratories), the sections were visualized using diaminobenzidine (DAB) detection reagent (Enzo Life Sciences, Farmingdale, NY, USA) and mounted with a mounting solution (Vector Laboratories, Burlingame, CA, USA).
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